Glycated proteins are contained in biological samples such as body fluid and hair, and body fluid includes blood in a living body, and such. The concentration of glycated proteins present in the blood depends on the concentration of sugars such as glucose dissolved in the serum, and recently, in the field of clinical diagnosis, measurement of the concentration of hemoglobin A1c (hereinafter, HbA1c; Non-Patent Document 1), which is a glycated protein in the blood, is being used to diagnose and monitor diabetes mellitus. As a method for measuring this HbA1c, instrumental analytical methods using high-performance liquid chromatography (HPLC) (Non-Patent Document 2), immunoassays using antigen-antibody reactions (for example, Non-Patent Document 3), and such had been known, but in recent years, enzymatic assays have been developed, and for example, a method using a protease and a glycated peptide oxidase (Patent Document 1) has been developed. Enzymatic assays can be applied to versatile automated analyzers, and since the operations are also simple, they are being developed actively.
The glycated peptide oxidase used in enzymatic assays is an enzyme that catalyzes the reaction which produces a sugar osone (an α-keto aldehyde), a peptide, and hydrogen peroxide by oxidatively cleaving, in the presence of oxygen molecules, the C—N bond in the ketose derivative produced by Amadori rearrangement of glucosylamine produced by the reaction between the hemiacetal of glucose and the N-terminal amino group of a peptide.
In the case of enzymatic assays, a method is known in which HbA1c is first degraded with a protease, and α-glycated valyl histidine (hereinafter, denoted as α-FVH) is produced from the N terminus of the β-chain of hemoglobin; next, glycated peptide oxidase is made to act on the produced α-FVH to produce hydrogen peroxide, a quinone dye is produced in the presence of peroxidase by the produced hydrogen peroxide, and the produced amount is determined by colorimetry using a spectrophotometer (Patent Document 1).
However, the glycated peptides to which conventionally-known glycated peptide oxidases (for example, Patent Documents 2, 3, 4, and 5, and Non-Patent Document 4) can act are ordinarily dipeptides, the largest being hexapeptides (Patent Documents 6 and 7), and glycated peptide oxidases that act on glycated peptides with longer amino acid chain length and on glycated proteins have not been known. Therefore, as described above, with regard to enzyme assays, the only method that has been reported is the method which comprises making a protease act on a glycated protein, making a glycated peptide oxidase act on the produced glycated peptide, and measuring the generated hydrogen peroxide.
With regard to this method which comprises making a protease act on glycated hemoglobin, making a glycated peptide oxidase act on the produced glycated peptide, and measuring the generated hydrogen peroxide, in case other measurements besides the glycated hemoglobin measurements are taken simultaneously using an automatic analyzer or such, the protease in the reagent for measuring glycated hemoglobin may act on other reagents and may affect the measured values.